This proposal is aimed at learning how DNA precursor biosynthesis is regulated in mammalian cells and how deoxyribonucleoside triphosphate (dNTP) production is coordinated with DNA replication and other cellular metabolic processes. In addition, we want to clarify the mechanism of action of antiproliferative agents which interfere with nucleic acid precursor biosynthesis. The proposed reearch involves: (1) Improvement and validation of analytical techniques for intracellular DNA precursor pools, including development of methods for dNTP assay; (2) characterization of DNA precursor compartmentation by determining the size and subcellular distribution of dNTP's in normal cells and after metabolic perturbations, by investigating preferential labeling of DNA by certain exogenous nucleosides, and by investigating the source and regulation of precursor supply for mitochondrial DNA; (3) studying the relationship of DNA precursor pools to metabolic events in herpes simplex virus infection; and (4) investigating the metabolic consequences of treating cultured cells with folate antagonists or fluorinated pyrimidines, particularly with respect to intracellular activity of thymidylate synthetase and nature of the DNA synthesized in inhibited cells.